Synchronization of Escherichia coli by zonal centrifugation.
A zonal centrifugation technique that can select the smallest newborn cells in an exponentially growing culture of Escherichia coli B/r is described.
Cell Division↗
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Purification and concentration of influenza inactivated viruses by continuous-flow zonal centrifugation.
A mathod is described for the purification, on an industrial scale, of influenza viruses grown in allantoic cavity of embryonated eggs. The mehtod consists of combining continuous-flow centrifugation with zonal centrifugation in a sucrose (36.6 per cent-52.5 per cent w/v) density gradient. The sample flow rate is approximately 3.7 litres/h and the volumes treated vary between 3 and 33 litres of allantoic fluid. Both the recovery of the virus and the degree of concentration and purification result satisfactory.
Allantois↗
Separation of Treponema pallidum from tissue substances by continuous-flow zonal centrifugation.
The zonal ultracentrifuge was used for separation of Treponema pallidum from large volumes of rabbit testicular syphiloma extracts by continuous-flow centrifugation in a cesium chloride density gradient. The gradient was linear with radius from a density of 1.05 to 1.36 g/ml. Operating speeds were 15,000 rev/min for the continuous-flow phase and 25,000 rev/min for a 30-min banding period. A total of 9 x 10(9) (24.3%) treponemes were recovered from the original extract. Of the treponemes recovered, 88% formed a band at a density of 1.170 to 1.211 g/ml. Within the limits of present methods of assay, these fractions were relatively free from testicular particulates and protein when compared with treponemes recovered after differential centrifugation. Observations of the isolated fractions by dark-field and electron microscopy indicated a lack of gross morphological damage to T. pallidum. Their antigenic characteristics were also retained, as evidenced by their ability to react with syphilitic sera in the indirect fluorescent-antibody procedure.
Animals↗
Lysosomes in rat thoracic duct lymphocytes fractionated by zonal centrifugation.
A method of zonal centrifugation was developed which separates rat thoracic duct lymphocytes (TDL) mainly according to size. The validity of the fractionation method was supported by light microscope observations, Coulter Counter sizing, and in vivo and in vitro labeling of lymphocytes. The distributions of lysosomal acid hydrolases in TDL fractionated by zonal centrifugation are similar to the distribution obtained for the cells. This result indicates that the large lymphocyte is not the sole bearer of either lysosomes or the large amount of soluble cathepsin D found in homogenates of TDL. Both reside mainly in small lymphocytes. This point was clearly established by fractionating homogenates of purified small lymphocytes by means of differential centrifugation and isopycnic density gradient centrifugation.
Acetamides↗
Lysosomes of cardiac and skeletal muscle: resolution by zonal centrifugation.
The application of zonal centrifugation to the analysis of homogenates of cardiac and skeletal muscle permits selection of fractions that are enriched in markers for lysosomes, sarcolemma, sarcoplasmic reticulum, and mitochondria. The method of disruption of normal and pathological tissue alters significantly the distribution of total protein and peaks of enzymatic activity on the gradient. Total activities of cathepsin, N-acetyl-beta-glucosaminidase, beta-glucuronidase, and para-nitrophenylphosphatase are distributed at different concentrations of sucrose on the gradient. Beta-Glucuronidase appears to "mark" the sarcoplasmic reticulum, as well as lysosomes, of skeletal muscle, para-Nitrophenylphosphatase, a common marker of acid phosphatase of lysosomes, is enriched in those fractions of cardiac muscle containing the highest specific activity of ouabain-inhibited Na-K-ATPase. Thus, these two enzymes appear to have a localization in at least two separate organelles. On the other hand, these results may indicate the isolation of several "populations" of lysosomes that are associated constantly with distribution peaks of other organelles. In any event, attempts to correlate changes in structure of organelles of normal and pathological specimens of tissue with functional impairment, e.g., Ca2+ uptake, activity of Na-K-ATPase, etc., must include consideration of dual localization of enzymatic markers or cross contamination by populations of other organelles.
4-Nitrophenylphosphatase↗
Immunological properties of Rickettsia rickettsii purified by zonal centrifugation.
The properties of Rickettsia rickettsii purified from infected chicken yolk sacs or mouse L cell cultures by sucrose density gradient centrifugation in a zonal rotor were examined in various ways. Rickettsiae derived from both L cells and yolk sacs had similar compositions: about 12% nitrogen, 1.5% phosphorus, 5% carbohydrate, and 30% fatty acids. On a dry-weight basis, purified rickettsiae were at least 2,000 times as effective as a commercial spotted fever vaccine in protecting guinea pigs against infection with spotted fever rickettsiae and mice against death from toxin of R. rickettsii. Gradient-purified rickettsiae (0.6 mug) induced a serological response in guinea pigs of the same magnitude as that stimulated by 1,600 mug of the commercial vaccine. Gradient-purified rickettsiae had little group reactivity in complement fixation tests but became anti-complementary upon storage. Microagglutination and hemagglutination tests with the purified antigen gave promise of usefulness in diagnosis of spotted fever. These results suggest that zonal centrifugation may be a valuable procedure for the preparation of R. rickettsii vaccine and diagnostic reagent.
Animals↗
ISOLATION OF RELAXING PARTICLES FROM RAT SKELETAL MUSCLES IN ZONAL CENTRIFUGES.
Supernatants of rat skeletal muscle homogenates were fractionated by differential centrifugation and by zonal centrifugation in sucrose density gradients. Cytochrome oxidase was employed as an enzymatic marker for locating mitochondria. The subcellular fractions were also assayed for their ability to prevent the ATP-induced contraction of myofibrils. Both the mitochondrial and microsomal fractions obtained by differential fractionation were found to be rich in such relaxing activity, and the microsomal fraction was appreciably contaminated by mitochondria. In contrast to this, when fractionation was carried out by means of zonal centrifugation (4200 RPM x 205 min. to 40,000 RPM x 60 min.), relaxing activity was found to be associated only with particles having the sedimentation characteristics of microsomes (s(20,w) estimated to be between 370 and 1880S). Relaxing activity was not detected in the regions of the gradient containing either the starting sample zone (soluble phase) or the mitochondrial peak. The microsomal relaxing particles showed negligible cytochrome oxidase activity.
Adenosine Triphosphate↗
Distribution of PNS myelin proteins and membrane enzymes in fractions isolated by continuous gradient zonal centrifugation.
Myelin was purified from adult rabbit sciatic nerve by two procedures: discontinuous gradient centrifugation and continuous gradient zonal centrifugation. Two fractions were obtained from the discontinuous gradient. The fraction floating on 0.32 M sucrose and the fraction recovered from the 0.32/0.85 M sucrose interface showed typical myelin membranes by electron microscopy and typical myelin proteins by gel electrophoresis. The specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) decreased from the top to the bottom of the discontinuous gradient. The myelin separated by zonal centrifugation on a continuous sucrose gradient showed three distinct peaks (on monitoring optical density) at 0.10, 0.30 and 0.57 M sucrose. The latter peak yielded 92% of the material applied. The two minor peaks of low density exhibited high CNP and acetylcholinesterase (AChE) activities but the specific activity of both enzymes increased markedly at the heavy end of the gradient. The zonal fractions showed typical myelin proteins in all fractions by polyacrylamide gel electrophoresis but with important quantitative differences. These results indicate that PNS myelin shows significant heterogeneity.
2',3'-Cyclic-Nucleotide Phosphodiesterases↗
The total containment of a batch-type zonal centrifuge.
A batch-type zonal centrifuge has been modified and totally contained for use with biologically hazardous materials. A sealed cabinet encloses the centrifuge and the ancilliary equipment. It is operated with a flow of filtered air when the zonal system is on, decontaminated with ethylene oxide, and maintained at a negative pressure throughout. The centrifuge subsystems can be drained, flushed, and decontaminated with ethylene oxide before an engineer services the machine. The sample handling system within the cabinet is remotely controlled.
Centrifugation, Zonal↗
HCO-3-ATPase activity distribution in rat liver cell fractions prepared by zonal centrifugation.
Plasma membrane sheets prepared by zonal centrifugation of a premicrosomal pellet obtained from a rat liver homogenate are devoid of HCO-3-ATPase activity. Since the microsomal fraction is also lacking in this ATPase activity, it can be concluded that the HCO-3-ATPase is not involved in the secretion of HCO-3 into bile.
Adenosine Triphosphatases↗
Use of yeast populations fractionated by zonal centrifugation to study the cell cycle.
Zonal centrifugation in a sucrose density gradient was used to separate yeast cells primarily by size and thus by age in the cell cycle. This approach provides an alternative to synchronous growth for examining the properties of cells at different stages in the cell cycle.
Cell Division↗
Large scale fractionation of calf brain by double run zonal centrifugation.
A previously described method for the subfractionation of subcellular particles by zonal centrifugation (Spanner, 1972), gives an unsatisfactory resolution when applied to calf brain, particularly when large amounts of subcellular particles are required. We succeeded in subfractionating 30-45 g of gray matter by modifying the procedure for preparing the P2 fraction (crude mitochondrial fraction) and introducing a new stepwise sucrose gradient on double run zonal centrifugation. Satisfactory resolution can also be achieved by single run zonal centrifugation when the starting material does not exceed 15 g of gray matter. The following fractions were separated as characterized chemically morphologically and by their enzyme markers: a) light myelin (0.4-0.6 M sucrose); b) heavy myelin (0.7 M); c) plasmamembranes (0.9-1.0 M); d) nerve endings (1.0-1.2 M); e) mitochondria (1.3 M); f) lysosomes (1.4 M).
Animals↗
Separation of abnormal cells in the peripheral blood by means of the zonal centrifuge.
The basic principles of rate centrifugation in the large volume zonal centrifuge and its application to the separation of abnormal or rarely occurring cells in the blood are described. Details of the method, gradient media, and collection and examination of the fractions obtained are given. A description of the cells found in normal human blood, in a subject undergoing known immunological stimulus, and in glandular fever and Hodgkin's disease is given and the implications are discussed.
Antigens↗
Variation in monoamine oxidase activity in rat brain crude mitochondrial fractions prepared by rate zonal centrifugation.
Rat brain crude mitochondrial fractionated by rate zonal centrifugation using an iso-osmotic gradient of Ficoll and sucrose. The results demonstrated that the isolated fractions were biochemically heterogeneous with regard to the enzymes, monoamine oxidase (MAO), NADH dehydrogenase and succinate dehydrogenase. When the activity of MAO was plotted as % of the highest specific activity towards tyramine, kynuramine oxidation remained fairly constant in fractions 10 to 30 but tyramine and dopamine showed separate peaks of activity in fractions 21 and 32 respectively. Sonic oscillations of separated particulate fractions did not change the ratios of various monoamine deamination when compared to the intact particles.
Animals↗
The fractionation of nuclei from mammalian cells by zonal centrifugation.
1. Purified liver nuclei from adult rats separate into two main zones when centrifuged in the slow-speed zonal rotor. One zone contains diploid nuclei, the other tetraploid. 2. The effect of age on the pattern of rat liver ploidy was examined. Tetraploid nuclei are virtually absent from young animals. They increase in proportion steadily with age. Partial hepatectomy disturbs the pattern of ploidy. 3. The zonal centrifuge permits the separation of diploid, tetraploid, octaploid and hexadecaploid nuclei from mouse liver. 4. Rat liver nuclei are isopycnic with sucrose solutions of density 1.35 at 5 degrees .
Age Factors↗
ANALYTICAL TECHNIQUES FOR CELL FRACTIONS. IV. REORIENTING GRADIENT ROTORS FOR ZONAL CENTRIFUGATION.
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Cell Biology↗
Purification of messenger ribonucleoprotein particles from rabbit reticulocytes by zonal centrifugation in metrizamide.
A large-scale purification procedure for messenger ribonucleoprotein (mRNP) particles from rabbit reticulocyte polysomes is described. The mRNP particles were dissociated from polysomes by treatment with urea and separated by differential centrifugation under conditions of high ionic strength. Zonal centrifugation in a metrizamide buoyant density gradient was the final purification step. One major class of mRNA particle was observed. The RNA was defined as mRNA by polyacrylamide gel electrophoresis and by globin production in a cell-free protein-synthesizing system. The twenty-three different proteins associated with the particle were a discrete set of proteins, which ranged in molecular weight from 175,000 to 23,500. The relative amount of each peptide in the particle was determined from a gel scan of the stained protein by computer simulation. None of the polypeptides comigrated with proteins from the 40-S and 60-S ribosomal subunits when analyzed by two-dimensional polyacrylamide gel electrophoresis.
Animals↗
Preparative zonal centrifugation.
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Animals↗